Project Summary Tight junctions (TJs) at the boundaries of endothelial and epithelial cells are critical in the development and function of vertebrates because they enable these tissues to separate, protect, and shape external epidermis and limbs and internal organs and glands. TJs regulate molecular transport through the spaces between individual cells (paracellular) while adhering cellular sheets. TJs perform two vital functions in tissues: 1) form barriers to restrict paracellular flux of small molecules, protecting organisms from the external environment and separating internal body compartments; and 2) creating size- and charge-selective pores, allowing permeability of ions that maintain electrochemical gradients. Numerous proteins amass at TJs to form the macromolecular assemblies necessary for barrier and pore function. But two families of membrane proteins?claudins and TAMPs (TJ-associated Marvel proteins)?predominate TJ assembly, architecture, and function. As these TJ integral membrane proteins (TJIMPs) are the sole components to span intracellular, intramembraneous, and extracellular space, they act as cytoskeletal scaffolds and assemble side-by-side within a membrane (cis) and with TJIMPs from adjacent cell membranes (trans) to form barriers and pores. The molecular structure of TJs is dynamic. Changes in protein composition, interaction, conformation, or modification?useful for assembling TJs to precisely tune paracellular transport under normal conditions?can also be mis-assembled, resulting in pathologies such as cancer, Alzheimer?s, Parkinson?s, Huntington?s, ALS, stroke, food poisoning and inflammatory bowel disease, renal wasting, hepatitis, and diseases of the skin, eyes, and ears. Molecular level insights into TJ structure and dynamics; the mechanisms of assembly that govern barrier and pore function; and how disabling these mechanisms leads to pathologies, remain unresolved matters in our fundamental understanding of TJs. We propose here a comprehensive research program that uses highly interdisciplinary approaches to determine structure?interaction?function relationships between TJIMPs at dynamic TJ microenvironments. These approaches integrate structural biology of TJIMPs and their complexes with information obtained by traditional and state-of-the-art bioinformatics, biochemical, biophysical, and functional experiments. The research program intends to resolve the underlying molecular principles of TJ assembly and disassembly by confronting technical challenges and, in the near-term, by answering specific questions on TJIMP interaction networks, the basis of gut barrier breakdown by a bacterial toxin, and the mechanisms of TJIMP form and function at the blood-brain barrier. The long-term goal of our laboratory is to elucidate the molecular bases for construction, destruction, and reconstruction of TJs, occurring both naturally or via disease-causing mechanisms, and to use the achieved insights to advance design and development of novel therapeutics to remedy TJ-related ailments.